Analysis of mutation status and homologous recombination deficiency in tumors of patients with germline BRCA1 or BRCA2 mutations and metastatic breast cancer: OlympiAD.

BACKGROUND: Presence of a germline BRCA1 and/or BRCA2 mutation (gBRCAm) may sensitize tumors to poly(ADP-ribose) polymerase (PARP) inhibition via inactivation of the second allele, resulting in gene-specific loss of heterozygosity (gsLOH) and homologous recombination deficiency (HRD). Here we explore whether tissue sample testing provides an additional route to germline testing to inform treatment selection for PARP inhibition. PATIENTS AND METHODS: In this prespecified exploratory analysis, BRCA1 and/or BRCA2 mutations in blood samples (gBRCAm) and tumor tissue (tBRCAm) were analyzed from patients with human epidermal growth factor receptor 2 (HER2)-negative metastatic breast cancer and known gBRCAm, enrolled in the phase III OlympiAD trial. The frequency and nature of tBRCAm, HRD score status [HRD-positive (score ≥42) versus HRD-negative (score <42) using the Myriad myChoice® CDx test] and rates of gsLOH were determined, and their impact on clinical efficacy (objective response rate and progression-free survival) was explored. RESULTS: Tissue samples from 161/302 patients yielded tBRCAm, HRD and gsLOH data for 143 (47%), 129 (43%) and 125 (41%) patients, respectively. Concordance between gBRCAm and tBRCAm was 99%. gsLOH was observed in 118/125 (94%) patients [BRCA1m, 73/76 (96%); BRCA2m, 45/49 (92%)]. A second mutation event was recorded for two of the three BRCA1m patients without gsLOH. The incidence of HRD-negative was 16% (21/129) and was more common for BRCA2m (versus BRCA1m) and/or for hormone receptor-positive (versus triple-negative) disease. Olaparib antitumor activity was observed irrespective of HRD score. CONCLUSIONS: gBRCAm identified in patients with HER2-negative metastatic breast cancer by germline testing in blood was also identified by tumor tissue testing. gsLOH was common, indicating a high rate of biallelic inactivation in metastatic breast cancer. Olaparib activity was seen regardless of gsLOH status or HRD score. Thus, additional tumor testing to inform PARP inhibitor treatment selection may not be supported for these patients.

Vistusertib (dual m-TORC1/2 inhibitor) in combination with paclitaxel in patients with high-grade serous ovarian and squamous non-small-cell lung cancer.

Background: We have previously shown that raised p-S6K levels correlate with resistance to chemotherapy in ovarian cancer. We hypothesised that inhibiting p-S6K signalling with the dual m-TORC1/2 inhibitor in patients receiving weekly paclitaxel could improve outcomes in such patients. Patients and methods: In dose escalation, weekly paclitaxel (80 mg/m2) was given 6/7 weeks in combination with two intermittent schedules of vistusertib (dosing starting on the day of paclitaxel): schedule A, vistusertib dosed bd for 3 consecutive days per week (3/7 days) and schedule B, vistusertib dosed bd for 2 consecutive days per week (2/7 days). After establishing a recommended phase II dose (RP2D), expansion cohorts in high-grade serous ovarian cancer (HGSOC) and squamous non-small-cell lung cancer (sqNSCLC) were explored in 25 and 40 patients, respectively. Results: The dose-escalation arms comprised 22 patients with advanced solid tumours. The dose-limiting toxicities were fatigue and mucositis in schedule A and rash in schedule B. On the basis of toxicity and pharmacokinetic (PK) and pharmacodynamic (PD) evaluations, the RP2D was established as 80 mg/m2 paclitaxel with 50 mg vistusertib bd 3/7 days for 6/7 weeks. In the HGSOC expansion, RECIST and GCIG CA125 response rates were 13/25 (52%) and 16/25 (64%), respectively, with median progression-free survival (mPFS) of 5.8 months (95% CI: 3.28-18.54). The RP2D was not well tolerated in the SqNSCLC expansion, but toxicities were manageable after the daily vistusertib dose was reduced to 25 mg bd for the following 23 patients. The RECIST response rate in this group was 8/23 (35%), and the mPFS was 5.8 months (95% CI: 2.76-21.25). Discussion: In this phase I trial, we report a highly active and well-tolerated combination of vistusertib, administered as an intermittent schedule with weekly paclitaxel, in patients with HGSOC and SqNSCLC. Clinical trial registration: ClinicialTrials.gov identifier: CNCT02193633.

The Effect of MHC Antigen Matching Between Donors and Recipients on Skin Tolerance of Vascularized Composite Allografts.

The emergence of skin-containing vascularized composite allografts (VCAs) has provided impetus to understand factors affecting rejection and tolerance of skin. VCA tolerance can be established in miniature swine across haploidentical MHC barriers using mixed chimerism. Because the deceased donor pool for VCAs does not permit MHC antigen matching, clinical VCAs are transplanted across varying MHC disparities. We investigated whether sharing of MHC class I or II antigens between donors and recipients influences VCA skin tolerance. Miniature swine were conditioned nonmyeloablatively and received hematopoietic stem cell transplants and VCAs across MHC class I (n = 3) or class II (n = 3) barriers. In vitro immune responsiveness was assessed, and VCA skin-resident leukocytes were characterized by flow cytometry. Stable mixed chimerism was established in all animals. MHC class II-mismatched chimeras were tolerant of VCAs. MHC class I-mismatched animals, however, rejected VCA skin, characterized by infiltration of recipient-type CD8+ lymphocytes. Systemic donor-specific nonresponsiveness was maintained, including after VCA rejection. This study shows that MHC antigen matching influences VCA skin rejection and suggests that local regulation of immune tolerance is critical in long-term acceptance of all VCA components. These results help elucidate novel mechanisms underlying skin tolerance and identify clinically relevant VCA tolerance strategies.

Deep brain stimulation in ireland for Parkinson's disease and essential tremor.

Deep brain stimulation (DBS) is highly effective neurosurgery for idiopathic Parkinson's disease (IPD), essential tremor (ET) and primary dystonia. DBS involves stereotactic surgical implantation of a battery-operated stimulator into deep brain nuclei. Irish patients are referred abroad for DBS and have to travel repeatedly for pre and post-operative care resulting in stress, anxiety and hardship. Safe pre and post-operative care of these complex, ageing patients is compromised by the absence of a DBS service in Ireland. Moreover, both DBS surgery and the subsequent post-operative care abroad incurs substantial cost to the state. The Dublin Neurological Institute at the Mater Misericordiae University Hospital (DNI) is a non-profit institute for the care of patients with neurological diseases. The DNI developed, in collaboration with the Mater Private Hospital (MPH) and the Walton Centre, Liverpool, a DBS programme in 2008/2009. We performed DBS at the Mater Campus on three carefully selected patients from a cohort of movement disorder patients attending the DNI and continue to provide pre-operative assessment and post operative care for patients following DBS in Ireland and abroad.

Role of protein tyrosine phosphatase SHP2 in barrier function of pulmonary endothelium.

Pulmonary edema is mediated in part by disruption of interendothelial cell contacts. Protein tyrosine phosphatases (PTP) have been shown to affect both cell-extracellular matrix and cell-cell junctions. The SH2 domain-containing nonreceptor PTP, SHP2, is involved in intercellular signaling through direct interaction with adherens junction proteins. In this study, we examined the role of SHP2 in pulmonary endothelial barrier function. Inhibition of SHP2 promoted edema formation in rat lungs and increased monolayer permeability in cultured lung endothelial cells. In addition, pulmonary endothelial cells demonstrated a decreased level of p190RhoGAP activity following inhibition of SHP2, events that were accompanied by a concomitant increase in RhoA activity. Furthermore, immunofluorescence microscopy confirmed enhanced actin stress fiber formation and diminished interendothelial staining of adherens junction complex-associated proteins upon SHP2 inhibition. Finally, immunoprecipitation and immunoblot analyses demonstrated increased tyrosine phosphorylation of VE-cadherin, beta-catenin, and p190RhoGAP proteins, as well as decreased association between p120-catenin and VE-cadherin proteins. Our findings suggest that SHP2 supports basal pulmonary endothelial barrier function by coordinating the tyrosine phosphorylation profile of VE-cadherin, beta-catenin, and p190RhoGAP and the activity of RhoA, signaling molecules important in adherens junction complex integrity.

Early surfactant administration with brief ventilation vs. selective surfactant and continued mechanical ventilation for preterm infants with or at risk for respiratory distress syndrome.

BACKGROUND: Both prophylactic and early surfactant replacement therapy reduce mortality and pulmonary complications in ventilated infants with respiratory distress syndrome (RDS) compared with later selective surfactant administration. However, continued post-surfactant intubation and ventilation are risk factors for bronchopulmonary dysplasia (BPD). The purpose of this review was to compare outcomes between two strategies of surfactant administration in infants with RDS; prophylactic or early surfactant administration followed by prompt extubation, compared with later, selective use of surfactant followed by continued mechanical ventilation. OBJECTIVES: To compare two treatment strategies in preterm infants with or at risk for RDS: early surfactant administration with brief mechanical ventilation (less than one hour) followed by extubation vs. later selective surfactant administration, continued mechanical ventilation, and extubation from low respiratory support. Two populations of infants receiving early surfactant were considered: spontaneously breathing infants with signs of RDS (who receive surfactant administration during evolution of RDS prior to requiring intubation for respiratory failure) and infants at high risk for RDS (who receive prophylactic surfactant administration within 15 minutes after birth). SEARCH STRATEGY: Searches were made of the Oxford Database of Perinatal Trials, MEDLINE (1966 - December 2006), CINAHL (1982 to December Week 2, 2006), EMBASE (1980 - December 2006), Cochrane Central Register of Controlled Trials (CENTRAL, The Cochrane Library, Issue 4, 2006), Pediatric Research (1990 - 2006), abstracts, expert informants and hand searching. No language restrictions were applied. SELECTION CRITERIA: Randomized or quasi-randomized controlled clinical trials comparing early surfactant administration with planned brief mechanical ventilation (less than one hour) followed by extubation vs. selective surfactant administration continued mechanical ventilation, and extubation from low respiratory support. DATA COLLECTION AND ANALYSIS: Data were sought regarding effects on the incidence of mechanical ventilation (ventilation continued or initiated beyond one hour after surfactant administration), incidence of bronchopulmonary dysplasia (BPD), chronic lung disease (CLD), mortality, duration of mechanical ventilation, duration of hospitalization, duration of oxygen therapy, duration of respiratory support (including CPAP and nasal cannula), number of patients receiving surfactant, number of surfactant doses administered per patient, incidence of air leak syndromes (pulmonary interstitial emphysema, pneumothorax), patent ductus arteriosus requiring treatment, pulmonary hemorrhage, and other complications of prematurity. Stratified analysis was performed according to inspired oxygen threshold for early intubation and surfactant administration in the treatment group: inspired oxygen within lower (FiO2< 0.45) or higher (FiO2 > 0.45) range at study entry. Treatment effect was expressed as relative risk (RR) and risk difference (RD) for categorical variables, and weighted mean difference (WMD) for continuous variables. MAIN RESULTS: Six randomized controlled clinical trials met selection criteria and were included in this review. In these studies of infants with signs and symptoms of RDS, intubation and early surfactant therapy followed by extubation to nasal CPAP (NCPAP) compared with later selective surfactant administration was associated with a lower incidence of mechanical ventilation [typical RR 0.67, 95% CI 0.57, 0.79], air leak syndromes [typical RR 0.52, 95% CI 0.28, 0.96] and BPD [typical RR 0.51, 95% CI 0.26, 0.99]. A larger proportion of infants in the early surfactant group received surfactant than in the selective surfactant group [typical RR 1.62, 95% CI 1.41, 1.86]. The number of surfactant doses per patient was significantly greater among patients randomized to the early surfactant group [WMD 0.57 doses per patient, 95% CI 0.44, 0.69]. In stratified analysis by FIO2 at study entry, a lower threshold for treatment (FIO2< 0.45) resulted in lower incidence of airleak [typical RR 0.46 and 95% CI 0.23, 0.93] and BPD [typical RR 0.43, 95% CI 0.20, 0.92]. A higher treatment threshold (FIO2 > 0.45) at study entry was associated with a higher incidence of patent ductus arteriosus requiring treatment [typical RR 2.15, 95% CI 1.09, 4.13]. AUTHORS' CONCLUSIONS: Early surfactant replacement therapy with extubation to NCPAP compared with later selective surfactant replacement and continued mechanical ventilation with extubation from low ventilator support is associated with less need mechanical ventilation, lower incidence of BPD and fewer air leak syndromes. A lower treatment threshold (FIO2< 0.45) confers greater advantage in reducing the incidences of airleak syndromes and BPD; moreover a higher treatment threshold (FIO2 at study > 0.45) was associated with increased risk of PDA. These data suggest that treatment with surfactant by transient intubation using a low treatment threshold (FIO2< 0.45) is preferable to later, selective surfactant therapy by transient intubation using a higher threshold for study entry (FIO2 > 0.45) or at the time of respiratory failure and initiation of mechanical ventilation.

Quantitative assessment of protein function prediction from metagenomics shotgun sequences.

To assess the potential of protein function prediction in environmental genomics data, we analyzed shotgun sequences from four diverse and complex habitats. Using homology searches as well as customized gene neighborhood methods that incorporate intergenic and evolutionary distances, we inferred specific functions for 76% of the 1.4 million predicted ORFs in these samples (83% when nonspecific functions are considered). Surprisingly, these fractions are only slightly smaller than the corresponding ones in completely sequenced genomes (83% and 86%, respectively, by using the same methodology) and considerably higher than previously thought. For as many as 75,448 ORFs (5% of the total), only neighborhood methods can assign functions, illustrated here by a previously undescribed gene associated with the well characterized heme biosynthesis operon and a potential transcription factor that might regulate a coupling between fatty acid biosynthesis and degradation. Our results further suggest that, although functions can be inferred for most proteins on earth, many functions remain to be discovered in numerous small, rare protein families.

Thermal changes and cure depths associated with a high intensity light activation unit.

OBJECTIVES: The objectives of this study were to evaluate the thermal emission and curing characteristics of a high intensity halogen light (Astralis 10-Ivoclar Vivadent, Schaan. Leichtenstein) alone and on curing a conventional and a fast-curing micro-hybrid composite. METHODS: A bead thermistor was placed in the base of a standard model cavity. The cavity was irradiated using the light unit whilst empty, and when filled with either composite. Temperature rises were recorded using the light in four different output modes. Further samples were prepared to assess depth of cure via a digital penetrometer, light transmission using a computer-based radiometer, and microhardness with a Wallace hardness tester. RESULTS: Mean peak temperature rises recorded during polymerisation of the composites ranged from 6.9 degrees C for the product InTen-S cured with the Adhesive programme (Adh) to 11.0 degrees C for the product Tetric Ceram HB cured with the High Power (HI P) programme. A significantly greater depth of cure was obtained for InTen-S in line with the greater light transmission obtained for this material. DISCUSSION: The very high thermal emission characteristics reported in a recently published investigation for this light unit were not confirmed. CONCLUSIONS: As the extent of thermal trauma that can be tolerated by the dental pulp is unknown consideration should be given to the choice of light activation unit and curing programme when polymerising light activated resin based restorations in deep cavities close to the pulp.

Co-registration of magnetoencephalography with magnetic resonance imaging using bite-bar-based fiducials and surface-matching.

OBJECTIVE: To introduce a new technique for co-registration of Magnetoencephalography (MEG) with magnetic resonance imaging (MRI). We compare the accuracy of a new bite-bar with fixed fiducials to a previous technique whereby fiducial coils were attached proximal to landmarks on the skull. METHODS: A bite-bar with fixed fiducial coils is used to determine the position of the head in the MEG co-ordinate system. Co-registration is performed by a surface-matching technique. The advantage of fixing the coils is that the co-ordinate system is not based upon arbitrary and operator dependent fiducial points that are attached to landmarks (e.g. nasion and the preauricular points), but rather on those that are permanently fixed in relation to the skull. RESULTS: As a consequence of minimizing coil movement during digitization, errors in localization of the coils are significantly reduced, as shown by a randomization test. Displacement of the bite-bar caused by removal and repositioning between MEG recordings is minimal ( approximately 0.5 mm), and dipole localization accuracy of a somatosensory mapping paradigm shows a repeatability of approximately 5 mm. The overall accuracy of the new procedure is greatly improved compared to the previous technique. CONCLUSIONS: The test-retest reliability and accuracy of target localization with the new design is superior to techniques that incorporate anatomical-based fiducial points or coils placed on the circumference of the head.

Rapid generation of thrombin by atheroma and platelets.

Rapid re-occlusion of an atheromatous vessel after angioplasty may occur through yet incompletely known mechanisms. Atheromatous plaque has been shown to contain tissue factor (TF) activity. When atheroma extracts (atheroma) and platelets are incubated together a powerful prothrombinase is rapidly generated, which neither platelets nor atheroma alone can generate. Large amounts of thrombin were generated in minutes by many atheroma-platelet mixtures. However in these mixtures, generation of factor (F)Xa activity was not enhanced, but was in fact decreased by platelet tissue factor pathway inhibitor (TFPI) activity. Leukocytes had no appreciable effect in these short-term experiments. Although levels of factor VII and FX in atheroma were extremely low, antibodies to each of these factors inhibited prothrombinase formation. So did an antibody to factor V. A FXa inhibitor, DX 9065a, was very effective in preventing prothrombinase generation. These findings may explain the rapid occlusion that has been observed after angioplasty and point to avenues of prevention.

Factors affecting friction in the pre-adjusted appliance.

A jig was constructed to measure the frictional forces created by various tip and torque values in association with two types of straightwire bracket moving along tainless steel (SS) archwires. Forces were measured during translation of the bracket using an Instron machine. Steel and cobalt chromium brackets were tested in association with 0.019 x 0.025 and 0.021 x 0.025 inch steel archwires at tips from 0 to 3 degrees and torque values in 2 degree increments from 0 to 6 degrees. The mean values for static (2.2 N) and kinetic (2.1 N) friction were very similar (P = 0.71), as were the overall friction values for stainless steel (2.1 N) and chromium cobalt (2.2 N) brackets of similar dimensions (P = 0.44). Use of 0.021 x 0.025 inch wire produced three times as much friction as 0.019 x 0.025 inch wire, 3.0 N against 1.2 N (P < 0.01). Increased tip and torque were associated with highly significant increases in friction (P < 0.01). Every degree of tip produced approximately twice as much friction as comparable torque. The main conclusion of the study was that space closure should be completed on a 0.019 x 0.025 inch archwire before a 0.021 x 0.025 inch wire is used to complete tooth alignment.

Cuspal movement and microleakage in premolar teeth restored with a packable composite cured in bulk or in increments.

INTRODUCTION: The aim of this study was to investigate the effect of two novel curing systems (a plasma arc light, and a 'turbo-boosted' conventional curing light) on cuspal movement and gingival microleakage of 'packable' resin-based composite (RBC) restorations placed in extracted maxillary premolar teeth. MATERIALS AND METHODS: Forty sound extracted upper premolar teeth were subjected to standardised preparation of a large mesio-occlusal-distal cavity before restoration with a RBC. Four curing regimens were used. Either the RBC was placed in bulk and light-cured in one increment using (a). the plasma arc light; (b). the 'turbo-boosted' curing light, or the RBC was placed in eight increments using (c). the plasma arc light; (d). the 'turbo-boosted' curing light. A deflection measuring gauge allowed a measurement of cuspal deflection at each stage of polymerisation. Restored teeth were thermocycled before immersion in a 0.2% basic fuchsin dye for 24 h. After sagittal sectioning of the restored teeth in a mesio-distal plane, the sectioned restorations were examined to assess cervical microleakage. RESULTS: Cuspal deflection measurements were significantly increased when the 'turbo-boosted' halogen curing light was compared with the plasma arc light. Total mean cuspal deflection measurements obtained with incremental cure were significantly increased compared with bulk cure for both light sources. Gingival microleakage for bulk restored teeth was significantly increased compared with teeth restored incrementally. Incremental restoration with the plasma arc light had significantly increased gingival microleakage compared with the 'turbo-boosted' halogen curing light. CONCLUSIONS: The packable composite tested could not be cured adequately to a depth of 5 mm with the plasma arc light within the specified irradiation time. Under the test conditions of the current investigation, bulk curing only appeared to be practical with the high intensity halogen light (40 s activation). Incremental build-up and polymerisation optimised marginal seal for the high intensity halogen light but led to greater cuspal deflection.

A pilot investigation of operator variability during intra-oral light curing.

OBJECTIVE: To test the hypothesis that operator experience influences the efficacy of light curing in a typical posterior intra-oral location. To investigate whether short cure cycles affect performance. DESIGN: A cross-sectional single-centre study designed to assess the efficacy of experienced and inexperienced operators when undertaking simulated intra-oral curing. SETTING: An in vitro laboratory based investigation conducted in a dental school during 2001. MATERIALS AND METHODS: A computer-based technique was used to monitor light intensity in a clinical simulation. Dentists and student operators were tested for their ability to cure a posterior restoration effectively. Relative light intensity was assessed against time for each operator and test run. RESULTS: Experienced (qualified) operators produced more effective and consistent cure results than less experienced undergraduate students. Operator performance was not affected by variations in irradiation time. CONCLUSIONS: This cross-sectional pilot investigation demonstrates that operator experience is a factor in successful clinical photo-curing of posterior restorations. Stable and accurate light guide positioning are required throughout the entire irradiation cycle to optimise intra-oral cure of light-activated restorations. Further investigations are planned to assess the potential of this novel method of assessment for use as a routine teaching aid in clinical practice.

M channel KCNQ2 subunits are localized to key sites for control of neuronal network oscillations and synchronization in mouse brain.

Mutations in the potassium channel subunit KCNQ2 lead to benign familial neonatal convulsions, a dominantly inherited form of generalized epilepsy. In heterologous cells, KCNQ2 expression yields voltage-gated potassium channels that activate slowly (tau, approximately 0.1 sec) at subthreshold membrane potentials. KCNQ2 associates with KCNQ3, a homolog, to form heteromeric channels responsible for the M current (I(M)) in superior cervical ganglion (SCG) neurons. Muscarinic acetylcholine and peptidergic receptors inhibit SCG I(M), causing slow EPSPs and enhancing excitability. Here, we use KCNQ2N antibodies, directed against a conserved N-terminal portion of the KCNQ2 polypeptide, to localize KCNQ2-containing channels throughout mouse brain. We show that KCNQ2N immunoreactivity, although widespread, is particularly concentrated at key sites for control of rhythmic neuronal activity and synchronization. In the basal ganglia, we find KCNQ2N immunoreactivity on somata of dopaminergic and parvalbumin (PV)-positive (presumed GABAergic) cells of the substantia nigra, cholinergic large aspiny neurons of the striatum, and GABAergic and cholinergic neurons of the globus pallidus. In the septum, GABAergic, purinergic, and cholinergic neurons that contribute to the septohippocampal and septohabenular pathways exhibit somatic KCNQ2 labeling. In the thalamus, GABAergic nucleus reticularis neurons that regulate thalamocortical oscillations show strong labeling. In the hippocampus, many PV-positive and additional PV-negative interneurons exhibit strong somatic staining, but labeling of pyramidal and dentate granule somata is weak. There is strong neuropil staining in many regions. In some instances, notably the hippocampal mossy fibers, evidence indicates this neuropil staining is presynaptic.

Effect of exposure intensity and post-cure temperature storage on hardness of contemporary photo-activated composites.

OBJECTIVES: The effect of variation in post-exposure storage temperature (18 vs. 37 degrees C) and light intensity (200 vs. 500mW/cm(2)) on micro-hardness of seven light-activated resin composite materials, cured with a Prismetics Mk II (Dentsply) light activation unit, were studied. METHODS: Hardness values at the upper and lower surfaces of 2mm thick disc shaped specimens of seven light-cured resin composite materials (Herculite XRV and Prodigy/Kerr, Z100 and Silux Plus/3M, TPH/Dentsply, Pertac-Hybrid/Espe, and Charisma/Kulzer), which had been stored dry, were determined 24h after irradiation with a Prismetics Mk II (Dentsply) light activation unit. RESULTS: Hardness values varied with product, surface, storage temperature, and curing light intensity. In no case did the hardness at the lower surface equal that of the upper surface, and the combination of 500mW/cm(2) intensity and 37 degrees C storage produced the best hardness results at the lower surface. CONCLUSIONS: Material composition had a significant influence on surface hardness. Only one of the seven products (TPH) produced a mean hardness values at the lower surface >80% of the maximum mean upper surface hardness obtained for the corresponding product at 500mW/cm(2) intensity/37 degrees C storage temperature when subjected to all four test regimes. Despite optimum post-cure storage conditions, 200mW/cm(2) intensity curing for 40s will not produce acceptable hardness at the lower surface of 2mm increments of the majority of products tested.

Relative merits of narrow versus standard diameter light guides.

The purpose of this study was to assess the relative effectiveness of narrow versus standard diameter light guides at curing the initial increment of composite in a deep cavity. The micro-hardness of 2 mm thick samples of a light activated microfilled posterior composite was determined. Composite was irradiated at 0, 1 or 6 mm light guide tip/composite surface distances. Light intensity was measured with a computer-based radiometer. Lower surface cure was reduced with increasing light guide tip distances. Narrow diameter guides offered no advantage over standard guides for polymerisation of the initial increment of composite in the base of a cavity.

Ataxia telangiectasia mutated is essential during adult neurogenesis.

Ataxia telangiectasia (A-T) is an autosomal recessive disease characterized by normal brain development followed by progressive neurodegeneration. The gene mutated in A-T (ATM) is a serine protein kinase implicated in cell cycle regulation and DNA repair. The role of ATM in the brain and the consequences of its loss on neuronal survival remain unclear. We studied the role of ATM in adult neural progenitor cells in vivo and in vitro to define the role of ATM in dividing and postmitotic neural cells from Atm-deficient (Atm(-/-)) mice in a physiologic context. We demonstrate that ATM is an abundant protein in dividing neural progenitor cells but is markedly down-regulated as cells differentiate. In the absence of ATM, neural progenitor cells of the dentate gyrus show abnormally high rates of proliferation and genomic instability. Atm(-/-) cells in vivo, and in cell culture, show a blunted response to environmental stimuli that promote neural progenitor cell proliferation, survival, and differentiation along a neuronal lineage. This study defines a role for ATM during the process of neurogenesis, demonstrates that ATM is required for normal cell fate determination and neuronal survival both in vitro and in vivo, and points to a mechanism for neuronal cell loss in progressive neurodegenerative diseases.

Protein tyrosine phosphatase-dependent proteolysis of focal adhesion complexes in endothelial cell apoptosis.

Adenosine and/or homocysteine causes endothelial cell apoptosis, a mechanism requiring protein tyrosine phosphatase (PTPase) activity. We investigated the role of focal adhesion contact disruption in adenosine-homocysteine endothelial cell apoptosis. Analysis of focal adhesion kinase (FAK), paxillin, and vinculin demonstrated disruption of focal adhesion complexes after 4 h of treatment with adenosine-homocysteine followed by caspase-induced proteolysis of FAK, paxillin, and p130(CAS). No significant changes were noted in tyrosine phosphorylation of FAK or paxillin. Pretreatment with the caspase inhibitor Z-Val-Ala-Asp-fluoromethylketone prevented adenosine-homocysteine-induced DNA fragmentation and FAK, paxillin, and p130(CAS) proteolysis. Asp-Glu-Val-Asp-ase activity was detectable in endothelial cells after 4 h of treatment with adenosine-homocysteine. The PTPase inhibitor sodium orthovanadate did not prevent endothelial cell retraction or FAK, paxillin, or vinculin redistribution. Sodium orthovanadate did block adenosine-homocysteine-induced FAK, paxillin, and p130(CAS) proteolysis and Asp-Glu-Val-Asp-ase activity. Thus disruption of focal adhesion contacts and caspase-induced degradation of focal adhesion contact proteins occurs in adenosine-homocysteine endothelial cell apoptosis. Focal adhesion contact disruption induced by adenosine-homocysteine is independent of PTPase or caspase activation. These studies demonstrate that disruption of focal adhesion contacts is an early, but not an irrevocable, event in endothelial cell apoptosis.

Adenosine induces endothelial apoptosis by activating protein tyrosine phosphatase: a possible role of p38alpha.

Endothelial cell (EC) apoptosis is important in vascular injury, repair, and angiogenesis. Homocysteine and/or adenosine exposure of ECs causes apoptosis. Elevated homocysteine or adenosine occurs in disease states such as homocysteinuria and tissue necrosis, respectively. We examined the intracellular signaling mechanisms involved in this pathway of EC apoptosis. Inhibition of protein tyrosine phosphatase (PTPase) attenuated homocysteine- and/or adenosine-induced apoptosis and completely blocked apoptosis induced by the inhibition of S-adenosylhomocysteine hydrolase with MDL-28842. Consistent with this finding, the tyrosine kinase inhibitor genistein enhanced apoptosis in adenosine-treated ECs. Adenosine significantly elevated the PTPase activity in the ECs. Mitogen-activated protein kinase activities were examined to identify possible downstream targets for the upregulated PTPase(s). Extracellular signal-regulated kinase (ERK) 1 activity was slightly elevated in adenosine-treated ECs, whereas ERK2, c-Jun NH(2)-terminal kinase-1, or p38beta activities differed little. The mitogen-activated protein kinase-1 inhibitor PD-98059 enhanced DNA fragmentation, suggesting that increased ERK1 activity is a result but not a cause of apoptosis in adenosine-treated ECs. Adenosine-treated ECs had diminished p38alpha activity compared with control cells; this effect was blunted on PTPase inhibition. These results indicate that PTPase(s) plays an integral role in the induction of EC apoptosis upon exposure to homocysteine and/or adenosine, possibly by the attenuation of p38alpha activity.

Endothelial proliferation, migration, and differentiation are blunted by conditionally expressed protein kinase C pseudosubstrate peptides.

Peptides based on the pseudosubstrate (PS) sequence of conventional protein kinase C isoenzymes (alpha, beta, gamma) specifically inhibit PKC activity in permeabilized cells, but whether PS can be used to study the role of PKC in the proliferation or migration of intact endothelial cells (EC) and angiogenesis is unknown. Peptides based on the PKCeta pseudosubstrate (etaPS) sequence were 3.5- to 8-fold more potent in inhibiting the PKCalpha, delta, epsilon, or eta kinase activity than was the peptide based on the PKCalpha pseudosubstrate (alphaPS) sequence. Thus, etaPS was conditionally overexpressed in intact EC and compared to alphaPS. Serum-induced growth of EC expressing etaPS was significantly slower than that of control EC. etaPS EC demonstrated slower rate of serum stimulated migration than that of either control or alphaPS EC. Expression of either etaPS or alphaPS produced slower rates of PMA induced EC migration, as compared to control EC. In an in vitro three-dimensional assay in which EC organize into capillary tubules, the EC that expressed etaPS formed fewer such tubules. This study shows that pseudosubstrate inhibitors derived from PKCeta are more potent both in vitro and in vivo than one based on the conventional isoenzyme PKCalpha. These data further support a role for PKC in proliferation and migration of intact EC, and angiogenesis.